Title:
The Expression, Purification, and Characterization of the Protein Gbr-22 Introduction:
The use of chromatography has been used for years in order to separate and purify different proteins [1]. This is extremely important because the protein can only be studied after this is done (be more specific) [2]. Proteins come in all different shapes and sizes. Because of the different methods are used to isolate different proteins (Not a complete sentence)[2]. In expression the protein is highlighted to stand out amongst the rest of the sample. The purpose of purification is to separate the highlighted protein from the rest of the sample. Finally characterization is used to show the different properties(What properties?) of the protein sample that was taken.
The purpose of this lab is to express, purify, and characterize the gbr-22 plasmid. It is believed that the protein will be transformed with the plasmid and change color.(Bacteria is transformed with the plasmid to make the protein) Materials & Methods:
In the expression part of the lab, the goal (goals are included in intro not M&M) was to have the protein (gbr-22) show up (wrong word choice/be more specific). In order to do this two transformation tubes were taken and filled(wrong word choice/be more specific)with BL21(DE3). The protein plasmid was added to one of the tubes, experimental. The tubes were then heat shocked for 45 seconds. After this, the material in the transformation plates were plated onto corresponding agar plates. These plates then incubated overnight. The next day, a starter culture was grown by adding ampicillin and LB broth to tubes. The bacteria with the plasmid in it was taken from the plates and put in the tubes. This culture was then moved to a flask and put in the shaking incubator until it turned purple, 2 days. After turning purple, bacterial was centrifuged and the purple pellet was left. After finding the weight of the pellet, the cells were re-suspended.(I see that you understand the overall procedure here but I think you need to work on wording it correctly/scientifically)
For the purification part of the lab, cyanase and lysozyme were added to purify(not to purify but to lyse bacteria) but to break the cell wall the purple part (the "purple part" is the protein)of the bacteria. After centrifuging, the supernatant was moved into a sterile tube and filtered through a syringe filter. A column and Ni-NTA was then used to purify the solution. The remaining protein was then washed with 2 imidazole solutions of different concentrations. The Nanodrop spectrophotometer was then used to measure the concentration at 280 and 574 wavelength.
For the final part of the lab, Characterization, the samples that were collected during purification were run through gel electrophoresis using SDS-page. After staining and drying the gel, the ladder was used to determine the molecular weight. Results:
Figure 1 No DNA Control (expression)
Figure 2 DNA Control (expression)
Figure 3 Fun Plate (expression)
Figure 4 Turbid Culture in Flask (expression)
Figure 5 Purple Protein Pellet (expression)
Figure 6 Protein Solution Reading at 280nm (purification)
Figure 7 Gel Electrophoresis Sample 1-6 and 4-6 in water (characterization)
Figure 8 Sample 1-6 and 4-6 out of water (characterization)
Figure 9 Molecular Weight Standard for Protein Ladder (characterization)
Discussion:
The lysozyme was used to break down the cell walls and release the protein, gbr-22. Cyanase was then added in order to break down the DNA and RNA from the protein. the hexa-histidine tag bound to the nickel which was attached to a large bead, by comparison. The nickel beads were used in the purification part of the lab because the bead that was attached to the nickel is very big and it keeps the protein in the column while everything else washed away. The six samples that were taken during purification were used during characterization. Sample one was taken after the cell wall were broken down by lysozyme. Sample two was taken after cyanase broke down the DNA and RNA. Sample three was taken after the lysate went through the column. Sample four was taken after the wash. Sample five was of the first elution and sample 6 was of the second elution. The estimate of the purity of the final protein was about 40%. (the discussion seems more like a methods section...I would try to analyze you results and what you think it meant) Conclusions:
In this three part lab, gbr-22 was first expressed, then purified and finally characterized using gel electrophoresis. For future labs and research these techniques could be used to find different proteins. Then check the molecular weight of the sample.
A source of error during the lab was shown during electrophoresis. Sample five and six showed multiply rings(bands) though only one should have been shown. This indicates that there must have been a contamination in those samples. Another source of error could have happened during the expression part of the lab. Some samples only took one day to turn purple in the shaking incubator. While others took longer. This may be because something was done wrong or there was more protein in those samples.(Hmm...I don't think this right) References: [1]"Protein Purification and Isolation." Protein Purification and Protein Isolation: Applications & Technologies: Life Science Research: Bio-Rad. N.p., n.d. Web. 17 Apr. 2013
[2]Lodish, Harvey. "Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions." Purifying, Detecting, and Characterizing Proteins. U.S. National Library of Medicine, 18 Dec. -0001. Web. 17 Apr. 2013.
The Expression, Purification, and Characterization of the Protein Gbr-22
Introduction:
The use of chromatography has been used for years in order to separate and purify different proteins [1]. This is extremely important because the protein can only be studied after this is done (be more specific) [2]. Proteins come in all different shapes and sizes. Because of the different methods are used to isolate different proteins (Not a complete sentence)[2]. In expression the protein is highlighted to stand out amongst the rest of the sample. The purpose of purification is to separate the highlighted protein from the rest of the sample. Finally characterization is used to show the different properties(What properties?) of the protein sample that was taken.
The purpose of this lab is to express, purify, and characterize the gbr-22 plasmid. It is believed that the protein will be transformed with the plasmid and change color.(Bacteria is transformed with the plasmid to make the protein)
Materials & Methods:
In the expression part of the lab, the goal (goals are included in intro not M&M) was to have the protein (gbr-22) show up (wrong word choice/be more specific). In order to do this two transformation tubes were taken and filled(wrong word choice/be more specific)with BL21(DE3). The protein plasmid was added to one of the tubes, experimental. The tubes were then heat shocked for 45 seconds. After this, the material in the transformation plates were plated onto corresponding agar plates. These plates then incubated overnight. The next day, a starter culture was grown by adding ampicillin and LB broth to tubes. The bacteria with the plasmid in it was taken from the plates and put in the tubes. This culture was then moved to a flask and put in the shaking incubator until it turned purple, 2 days. After turning purple, bacterial was centrifuged and the purple pellet was left. After finding the weight of the pellet, the cells were re-suspended.(I see that you understand the overall procedure here but I think you need to work on wording it correctly/scientifically)
For the purification part of the lab, cyanase and lysozyme were added to purify(not to purify but to lyse bacteria) but to break the cell wall the purple part (the "purple part" is the protein)of the bacteria. After centrifuging, the supernatant was moved into a sterile tube and filtered through a syringe filter. A column and Ni-NTA was then used to purify the solution. The remaining protein was then washed with 2 imidazole solutions of different concentrations. The Nanodrop spectrophotometer was then used to measure the concentration at 280 and 574 wavelength.
For the final part of the lab, Characterization, the samples that were collected during purification were run through gel electrophoresis using SDS-page. After staining and drying the gel, the ladder was used to determine the molecular weight.
Results:
Discussion:
The lysozyme was used to break down the cell walls and release the protein, gbr-22. Cyanase was then added in order to break down the DNA and RNA from the protein. the hexa-histidine tag bound to the nickel which was attached to a large bead, by comparison. The nickel beads were used in the purification part of the lab because the bead that was attached to the nickel is very big and it keeps the protein in the column while everything else washed away. The six samples that were taken during purification were used during characterization. Sample one was taken after the cell wall were broken down by lysozyme. Sample two was taken after cyanase broke down the DNA and RNA. Sample three was taken after the lysate went through the column. Sample four was taken after the wash. Sample five was of the first elution and sample 6 was of the second elution. The estimate of the purity of the final protein was about 40%. (the discussion seems more like a methods section...I would try to analyze you results and what you think it meant)
Conclusions:
In this three part lab, gbr-22 was first expressed, then purified and finally characterized using gel electrophoresis. For future labs and research these techniques could be used to find different proteins. Then check the molecular weight of the sample.
A source of error during the lab was shown during electrophoresis. Sample five and six showed multiply rings(bands) though only one should have been shown. This indicates that there must have been a contamination in those samples. Another source of error could have happened during the expression part of the lab. Some samples only took one day to turn purple in the shaking incubator. While others took longer. This may be because something was done wrong or there was more protein in those samples.(Hmm...I don't think this right)
References:
[1]"Protein Purification and Isolation." Protein Purification and Protein Isolation: Applications & Technologies: Life Science Research: Bio-Rad. N.p., n.d. Web. 17 Apr. 2013
[2]Lodish, Harvey. "Proteins Can Be Removed from Membranes by Detergents or High-Salt Solutions." Purifying, Detecting, and Characterizing Proteins. U.S. National Library of Medicine, 18 Dec. -0001. Web. 17 Apr. 2013.